Antibacterial Activity of Eight Medicinal Plants from the Traditional Pharmacopoeia of Niger

The emergence of multidrug bacterial resistance poses a great public health problem and requires a constant search for new antibacterial agents. However, Niger's flora possesses several medicinal plants used in traditional medicine to cure infectious diseases and can be used as sources of bioactive ingredients. This current study was designed to evaluate the antibacterial activity of eight plants used in the traditional pharmacopeia of Niger. The extracts were prepared by maceration using ethanol, methanol, and distilled water. The obtained extracts were screened against Salmonella spp., Shigella spp., and Escherichia coli using the microdilution method coupled with a resazurin-based assay. Phytochemical screening was performed using colorimetry, while the quantification of total polyphenols, total flavonoids, and total tannins was determined by spectrophotometry. Out of the eight plants obtained, five named Cassia italica, Limeum pterocarpum, Phyllanthus pentandrus, Strychnos innocua, and Ximenia americanum exhibited antibacterial activity with MICs ranging from 500 μg/mL to 2000 μg/mL. Phytochemical screening showed the presence of alkaloids, saponosides, tannins, flavonoids, terpenes/sterols, quinones, and polyphenols. The ethanolic and methanolic extracts of X. americana contained important quantities of total polyphenols, with 43.59 ± 0.15 and 41.97 ± 0.02 mg EAG/100 mg of extract, respectively. These extracts showed the highest contents of total tannins at 46.49 g/L and 45.52 g/L, respectively. For total flavonoids, the highest content was obtained with the methanolic extract of P. pentandrus, with 3.12 ± 0.01 mg QE/100 mg of extract. These findings justify the uses of these plants in traditional medicine for the treatment of infectious diseases such as diarrhea and can be used as starting points for the development of phytodrugs against infectious diarrhea.


Introduction
Infectious diseases are pathologies caused by microorganisms such as fungi, viruses, and bacteria. Te latter is responsible for most of the pathology, including diarrhea. Diarrhea is mainly caused by bacteria and viruses. Since the vaccine against rotavirus was approved, bacteria have become the leading cause of diarrhea [1] and are classifed as the second most common cause of death in children under fve years of age, with approximately 525,000 deaths each year [2]. Shigella spp., Salmonella spp., and E. coli are the principal bacteria responsible for diarrhea. In Niger, Shigella is the leading etiology of diarrhea, with 60.5% of episodes of severe shigellosis occurring mainly in children under 5 years of age [3]. Globally, the overwhelming majority of cases have occurred in developing countries, where poor sanitation and limited access to clean water facilitate the spread of enteric pathogens. Malnutrition and lack of proper care are major contributors to the high mortality rate, particularly among young children [4]. Te treatment of diarrhea is mainly based on the administration of antibiotics. For this reason, the WHO recommends the use of azithromycin and antibiotics from the fuoroquinolone family, particularly ciprofoxacin, as the frst line of therapy [5]. Unfortunately, the emergence of multidrug-resistant bacterial strains and the failure of this treatment led the WHO to include these bacteria on the list of 12 priority bacteria for which the search for new drugs is urgently needed [6]. Regarding the history of traditional medicine, interest in herbal medicines continues to increase. Indeed, many patients are increasingly practicing herbal self-medication [7]. Te fora of Niger is still rich and unexplored, and exploring these plants based on traditional uses can be the best avenue to search for new drugs against infectious diarrhea and to provide the population with efective new drugs without harmful efects and at a low cost. Tese results will make it possible to obtain scientifc data that validate the uses of these plants in traditional medicine. Te objective of this research is to evaluate the antibacterial activities of some medicinal plants of the traditional pharmacopoeia of Niger.

Plant Material and Extract Preparation.
Using information gathered from traditional uses, some plants were selected. Diferent plant parts from each selected plant were harvested in September 2020 in Kollo (Tillabery). Small specimens were taken for the department of biology, and their scientifc names were provided by the department of biology by comparison to the specimen deposit under the number of herbariums. Once in the laboratory, the plant parts were washed to remove all dirt, cut, and dried at room temperature for two weeks. After that, the plants were ground into a fne powder and kept for further study. Traditionally, the solvent most commonly used is water. In order to justify scientifcally the activity of these plants, an aqueous extract was performed for each plant. Organic extracts were chosen to see the efect of the extraction solvent on the activity. Te extracts were prepared by maceration using two organic solvents (ethanol and methanol) and distilled water. For this, 100 g of powder was soaked in 1000 mL of each solvent. Te solutions were stirred twice each day for 3 days. At the end of this period, the extracts were fltered using hydrophilic cotton, and the fltrates were concentrated at 40°C under pressure of 175 mbar for the ethanolic extract and 337 mbar for the methanolic extract using rotavapor (Heidolph). Te crude extracts were dried under ventilation at room temperature to remove the remaining solvent and kept at 4°C for further studies. and then diluted to 10 6 CFU/mL for the tests.

Determination of Minimum Inhibitory Concentrations.
Te minimum inhibitory concentrations were determined using the broth microdilution method, as described by CLSI in 2012 (protocol M09 A7) coupled with a resazurin-based assay [8]. For this, 6 serial dilutions of each extract and positive control (Amoxicillin) were performed to obtain diferent concentrations ranging from 2000 to 31.25 μg/mL and from 0.5 to 0.0071 μg/mL, respectively. Te fnal volume in each well was 200 μL and the fnal concentration of DMSO was less than 1% with no efect on bacteria growth. Te test was performed in Muller-Hinton broth (Sigma-Aldrich). Te fnal concentration of the bacterial suspension was 5 × 10 5 CFU/mL. Te negative control was made up with culture media and bacteria suspension, while the sterility control was made up with culture media alone. Te plates were covered and incubated at 37°C for 24 hrs. At the end of the incubation period, 20 μL of freshly prepared resazurin (0.15 mg/mL Sigma-Aldrich) was introduced into each well, followed by incubation in the dark for 30 min. At the end of the incubation time, the MIC was defned as the smallest concentration of plant crude extract at which there was no change in coloration from blue to pink, corresponding to the lack of visible bacterial growth.

Phytochemical Screening of Active Extracts.
Te diferent groups of secondary metabolites of active extracts were highlighted using colorimetry methods as described by Ciulei in 1982, Wagner andBladt in 1996, andHamid et al., in 2018, with minor modifcations regarding the solvent of solubilization of the crude extract [9][10][11]. Tannins and polyphenols were identifed by the FeCl 3 test, favonoids by the cyanidin reaction, saponosides by the foam test, quinones by the Bornträger test, triterpenes and steroids by the Liebermann-Burchard test, and alkaloids by the Dragendorf test.
2.5. Total Phenolics Quantifcation. Following the procedure outlined by Singleton et al., 1999 [12], the total phenolic contents were determined. In fact, the extracts were dissolved in DMSO at a concentration of 100 mg/mL and then diluted to a fnal concentration of 1 mg/mL in distilled water. Te Folin-Ciocalteu reagent (0.2 N, TECNO PHARM-CHEM HARYANA) was added to each test tube corresponding to each dilution that contained 0.5 mL of the solutions obtained after dilution, 2.5 mL of the reagent had been diluted 10 times, and the test tubes were then allowed to sit at room temperature for 5 minutes. Following the incubation time, 2 mL of a sodium carbonate solution (75 mg/ mL) was added, and the mixture was then agitated. Te test tubes were once again incubated for two hours at room temperature in the dark. Te etalon for the calibration curve was made up using gallic at diferent concentrations of 0, 20, 40, 60, 80, and 100 mg/L. Te optical density was obtained by a spectrophotometer (Termo Scientifc Evolution 300) at 760 nm against the blank (0.5 mL of Folin-Ciocalteu reagent and 0.5 mL of sodium carbonate). Te test was carried out twice, and the outcome was represented as mg of gallic acid (Sigma-Aldrich) equivalents per 100 mg of extract (mg GAE/ 100 mg extract) [13].

Total Flavonoid Quantifcation. Te method outlined by
Arvouet-Grand et al. in 1994 [14] was used to determine the total favonoid content. In fact, 2 mL of each extract were mixed with 2 mL of aluminum trichloride (AlCl 3 , Sigma-Aldrich) in methanol (2%), and the mixture was then left to sit at room temperature for 10 min in the dark. Te presence of favonoids was established by the yellow stable coloring that corresponded to the production of a favonoids-(Al 3+ ; 3Cl − ) complex in comparison to quercetin, which was quantifed spectrophotometrically at 415 nm (Termo Scientifc Evolution 300). Based on a linear calibration curve made up with quercetin (Sigma-Aldrich) concentrations ranging from 0 to 100 mg/L, the total favonoid levels in extracts were determined and expressed in mg quercetin equivalents (mg QE)/100 mg of dry extract [13].

Total Tannin Quantifcation.
According to Ribereau-Gayon and Stonestreet in 1966 and Bate-Smith et al. in 1965 [15,16], the reaction of Bate-Smith was used to determine the tannin content. Tree (3 mL) mL of hydrochloric acid (12 N) were added to 2 mL of each plant's aqueous extract (1 mg/mL) in a hydrolysis tube (glass tube). Te tube was then sealed with a Tefon-sealed stopper and heated in a water bath to 100°C for 30 minutes. A control tube with the identical solution inside was kept at room temperature in parallel. After the hydrolyzed tube had cooled, a spectrophotometer was used to measure the optical density at 550 nm (Termo Scientifc Evolution 300). Te optical densities were used to calculate the total tannin contents and expressing the results in g/L [13]. Te following formula was used to calculate the total tannin content: C � 19.33 (Doh-Dot) where C is the content of total tannins expressed in g/L, Doh is the optical density of the hydrolyzed tube, and Dot is the optical density of the control tube.

Plant Research and Extraction
Yields. According to traditional medicine, eight herbs from eight families and eight genera are used to treat diarrhea, stomachaches, typhoid fever, gonorrhea, and malaria. Te following plant species and their Herbarium number are listed in Table 1. Plants used to treat infectious diseases were identifed. Tose that are more widely used and have not been studied were selected. Te diferent parts (stem bark, leaves, leafy stems, and whole plants) of these plants were harvested for the extract preparation. Tese plants are widely used in traditional pharmacopeia in Niger. Te traditional healer from Niger mainly used water for the remedies' preparation [17][18][19][20][21]. Tree solvents were used to prepare 24 extracts, with extraction yields varying from 2.4 to 32.8 percent (Table 2). Te extraction yields were dependent on the type of plant, the parts employed, and the extraction solvent. Methanol extraction produced the maximum yield (32.8%) from the stem bark of X. americana, followed by water (32.0%) and ethanol (31.2%). In addition, using water as the extraction solvent, the leaves of Boscia senegalensis produced a signifcant yield of 27.5% (Table 2). Tis fnding showed that water and methanol are the best extraction solvents.

Minimum Inhibitory Concentration.
Te 24 obtained extracts were tested for their antibacterial activity, and the fndings are grouped in Table 2. Out of the 24 extracts, 11 (40.74%) from 5 diferent plant species had antibacterial activity, with MICs varying from 500 μg/mL to 2000 μg/mL. Te activity was bacterial and plant extract dependent. Te most efective extracts reported came from P. pentandrus. With a MIC of 1000 μg/mL, PPME exhibited anti-Shigella (S. fexneri, S. sonnei, and S. dysenteria) activity, whilst PPAQ at the same dose reduced the growth of S. fexneri and S. sonnei and at 2000 μg/mL inhibited S. dysenteria, and PPET at 2000 μg/mL was active against S. sonnei. Te other plant that inhibited Shigella spp. was X. americana, and the MIC of most of the extracts from this plant was 2000 μg/mL. According to Table 3, the clinical isolate S. typhi was the most susceptible to the investigated extracts, with MICs of 1000 μg/mL (CIME, BLME, and BLET) and 2000 μg/mL (LPME, PPME, and PPET), while PPAQ showed exhibited activity with the MIC of 1000 μg/mL against the clinical isolate of Salmonella typhimurium.

Qualitative Phytochemical Screening of Active Extracts.
A phytochemical screening was conducted, and the fndings are shown in Table 3, to identify the major phytochemical groups in the active extracts that may be accountable for the activity attained. Te table suggests that all extracts are high in polyphenols, terpene/sterol, favonoids, and tannins. Alkaloids are mainly found in P. pentandrus. Saponosides were discovered in the methanolic, aqueous, and X. americana, P. pentandrus, and B. linariifolia extracts. Except for the methanolic and ethanolic extracts from S. innocua, quinone was present in all extracts (Table 4).
All active extracts contained tannins, favonoids, and phenolic substances. Tese substances are referred to as antimicrobial agents. Teir quantity was calculated using the regression equation derived from the calibration curves because the activity can occasionally be concentration dependent.

Total Phenolic Compound Content of Plant Extracts.
Gallic acid was used as a standard to obtain the regression equation for the phenolic compound contents, which was y � 0.0078x + 0.0803 and R 2 � 0.9822 (Figure 1). From this equation, the amount of phenolic compounds in each extract was deducted, and they were then expressed as gallic acid equivalents per 100 mg of dry extract (Table 5). According to Table 5, they range from 8.97 ± 0.09 to 43.59 ± 0.15 mg GAE/ 100 mg of extract for phenolic components. Te highest amount of total phenolic compounds is found in the XAET and XAME extracts, which had 43.59 and 41.97 mg EAG/ 100 mg, respectively, and were followed by PPET and PPME extracts, which had 29.59 and 27.75 mg EAG/100 mg, respectively.

Total Flavonoid Content of Plant Extracts.
Te total favonoid contents were calculated using the regression curve ( Figure 2) obtained using quercetin as a standard and the regression equation y � 0.0294x + 0.0271 and R 2 � 0.99. Te total favonoid contents were expressed in milligrams of quercetin equivalent per hundred milligrams of dry extract (mg EQ/100 mg). Te obtained total favonoid concentrations ranged from 0.46 EQ/100 mg to 3.12 ± 0.01 EQ/100 mg. Te methanolic extract from P. pentandrus had the highest total favonoid concentration, and it was followed by the methanolic extract from Blepharis linariifolia (2.970 mg EQ/ 100 mg).

Total Tannin Content of Plant Extracts.
Te concentrations of total tannins ranged from 46.49 g/L to 0.36 g/L. With concentrations of 46.49 g/L and 45.52 g/L, respectively, the extracts XAET and XAME displayed the best contents. Te total tannin levels of the other extracts were extremely low. Te BLET extract showed the lowest content. Te extracts that contained the most total tannin did not have the best antibacterial efects. Te most efective extracts on the E. coli strain are the BLME, PPAQ, and SIME extracts, but they have low quantities of total tannins. Tese extracts' activity would be related to additional chemical groupings.

Discussion
Bacteria are the primary cause of diarrhea, which is the second highest cause of death for children under 5 in low-and middle-income nations. Te administration of antibiotics is the mainstay of diarrhea therapy. Sadly, the rise of multidrugresistant bacteria has resulted in treatment failure and keeps raising the mortality toll globally. It is thus vital to carry on looking for fresh substitute agents. Because of this, it has been discovered that some compounds from medicinal plants used in traditional medicine are efective against bacteria pathogens. Te goal of the current study was to evaluate the antibacterial properties of several therapeutic plants utilized in Niger's traditional pharmacopeia. According to traditional medicine, eight (8) plants from eight diferent plant families were employed by traditional healers. Tree solvents were used to prepare 24 extracts, which were then tested for antibacterial activity. Of them, 11 showed antibacterial activity with MICs ranging from 2000 to 500 μg/mL. Te threshold established by Tamokou et al., 2017 (highly active: MIC ≤100 μg/mL, signifcantly active: 100 < MIC ≤ 512 μg/ mL, moderately active: 512 < MIC ≤ 1024 μg/mL, low activity: 1024 < MIC ≤ 2048 g/mL, and considered not active: MIC > 2048 mg/mL) showed that the activity of extracts ranged from low active to moderately active [22]. Tese activities vary from one bacterial strain to another and from one extract to another. All extracts tested were inactive against S. enteridis, and the aqueous extract of P. pentandrus showed moderate activity against S. typhimurium with an MIC of 1000 μg/mL. For E. coli, the methanolic extracts of P. pentandrus, S. innocua, and B. linariifolia had good    --1000  -----CIAQ  --------CIET  --------LPME  --2000  -----LPAQ  --------LPET  --------PPME  --2000  1000  1000  1000  1000  500  PPAQ  -1000  -1000  1000 2000   [24]. All tested extracts were positive for polyphenols, favonoids, tannins, and terpenes/   Journal of Tropical Medicine sterols, and these diferent groups of secondary metabolites are important for their various biological activities. Ethanolic and methanolic extracts of Strychnos innocua showed the same phytochemical profle but diferent antibacterial activity profles. Te ethanolic extract of the same plant did not show any activity against the E. coli strain, but its methanolic extract showed an MIC of 500 μg/mL, which would be related to the diference in polarity between the two solvents, with methanol being the most polar. Tese activities would be linked to the presence of other classes of secondary metabolites. Flavonoids, sterols, and terpenes are classes of secondary metabolites known to have antiplasmodial and antibacterial activities; their presence in the extracts of these plants would explain their evaluated biological activities. Tannins have antidiarrheal properties and are enzyme inhibitors, and favonoids have anti-infammatory and antioxidant properties [25]. Te synergistic action between antidiarrheal and antiinfammatory properties and inhibition of the development of certain microorganisms is of paramount importance in the fght against certain infectious diseases [25]. Te presence of these two groups in the tested extracts should be noted.
Tis result demonstrated the importance of alkaloid and phenolic compounds in the antibacterial activity. In fact, phenolic compounds act on bacteria by modifying the permeability of cell membranes, the changes in various intracellular functions induced by hydrogen binding of the phenolic compounds to enzymes, or by the modifcation of the cell wall rigidity with integrity losses due to diferent interactions with the cell membrane [26]. Alkaloids inhibit bacterial growth through a variety of mechanisms, including inhibition of bacterial nucleic acid and protein synthesis, modifcation of bacterial cell membrane permeability, damage to the cell membrane and cell wall, inhibition of bacterial metabolism, and inhibition of efux pumps [27]. Flavonoids are able to link to soluble proteins that are present outside of the cells and with bacterial cell walls, thus promoting the formation of complexes [26,28]. Flavonoids may also act on bacteria by inhibiting both energy metabolism and DNA synthesis, thus afecting protein and RNA synthesis [29]. Te mechanism of action of tannins is explained by their ability to pass through the cell wall up to the internal membrane of the bacteria, their interference with the metabolism of the cell, and as a result, their destruction. It has been shown that tannic acid inhibits bacterial attachment to surfaces [30].
As the antibacterial activity is concentration dependent, the most reported ingredients were quantifed using spectrophotometry approaches. Ethanolic and methanolic extracts of X. americana contained the most important quantity of phenolic compound (43.59 ± 0.15 and 41.97 ± 0.02 mg EAG/100 mg dry extract, respectively). Tese same extracts of this plant showed the highest contents of total tannins. For total favonoids, the highest content was obtained in the methanolic extract of P. pentandrus with 3.12 ± 0.01 mg QE/100 mg dry extract, followed by the methanol extract of B. linariifolia. Te extracts BLET, BLME, SIME, and SIET are the most active against E. coli; however, these extracts are not the richest in polyphenols. Te activities in these extracts could be related to other classes of compounds or to the diversity of the structures of the phenolic compounds. Low contents of total phenolic compounds were obtained with the SIET and SIME extracts. Te extract SIME showed activity against the E. coli strain with an MIC of 500 μg/mL. Te PPME extract of this plant contained 3.12 mg QE/100 mg, and this extract showed an MIC of 500 µg/mL against the E. coli strain. Te PPME extract was followed by the BLME and BLET extracts with 2.97 and 2.82 mg EQ/100 mg, respectively; the BLME extract also showed activity against E. coli with an MIC of 500 μg/ mL. Te lowest contents of total favonoids were observed in the XAET and XAME extracts, with 0.49 and 0.46 mg EQ/ 100 mg, respectively. Te SIME extract shows an activity of 500 μg/mL on the E. coli ATCC strain but has a low total favonoid content of 1.82 mg EQ/100 mg. Flavonoids are a class of secondary metabolites that have key roles in signaling between plants and microbes, in defense as antimicrobial agents and feeding deterrents, and in UV protection [31]. Tese compounds are found in many plant genera and all parts of plants. Tey exhibit several biological efects, such as anti-infammatory, antimicrobial, antiviral, antiulcer, hepatoprotective, antitumor, and antioxidant activities [32]. Many biological properties of favonoids may be related to their capacity to penetrate into cell membranes and thus afect their biological activity. Isolated favonoids from Dorstenia angusticornis showed good antibacterial activity against some bacterial strains. Bacteriocin C shows good antibacterial activity against Escherichia coli with an MIC of 1.22 μg/mL and against Salmonella typhi with an MIC of 78.12 μg/mL. Angusticornin B has good antibacterial activity against Escherichia coli with MICs of 0.61 μg/mL, 19.53 μg/mL against Salmonella typhi, and 0.61 μg/mL against Shigella dysenteriae. Stipulin also shows good antibacterial activity against Shigella dysenteriae, with MICs of 19.53 μg/mL and 78.12 μg/mL against Salmonella typhi [33]. Te presence of favonoids in some extracts could justify the activity of these extracts.

Conclusion
Five (Cassia italica, Limeum pterocarpum, Phyllanthus pentandrus, Strychnos innocua, and Ximenia Americanum) of the eight plants showed antibacterial activity, and phytochemical analysis showed the presence of polyphenols, favonoids, tannins, sterols/terpenes, alkaloids, and quinone in the active extracts. Tese extracts contain important quantities of total favonoids, total tannins, and total polyphenols. All these fndings could justify the use of these plants in traditional medicine to cure infectious diseases such as diarrhea and demonstrate that these extracts could contain bioactive compounds. Ten, further study will be conducted following UHPLC-MS/MS guided isolation to isolate the diferent unknown compounds responsible for the activity obtained.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon request. 8 Journal of Tropical Medicine